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DETECTION AND DISCRIMINATION OF TARGET DNA SEQUENCE AND SINGLE NUCLEOTIDE
POLYMORPHISM DIRECTLY ON DOUBLE-STRANDED OLIGONUCLEOTIDE AND PCR PRODUCT
WITHOUT DENATURATION OF DSDNA SAMPLE BASED ON PNA-DSDNA HYBRID AND
UTILIZING PNA PROBE

Abstract

A method of detection and discrimination of target DNA sequences and of
single nucleotide polymorphisms (SNPs) directly on double-stranded DNA
(dsDNA) samples without the need for denaturation of the target dsDNAs.
This is achieved by the use of a PNA chain as a probe. This probe is
self-assembled on a gold electrode; hybridization of the probe with the
target dsDNA forms a PNA-dsDNA hybrid. The hybrid is then labeled with a
mediator, and the PNA-dsDNA hybridization is monitored. Monitoring of the
hybrid formation can be achieved using an electrochemical approach. This
method is able to detect and discriminate target DNA sequences and SNPs
on ds-oligonucleotides. Furthermore, this method is able to detect and
discriminate target DNA sequences and SNPs on double-stranded PCR
products.

1. A PNA-based method for the detection and discrimination of target DNA
sequences directly on ds-oligonucleotides and double-stranded PCR
products, said method comprising products, said method comprising a.
creating a PNA probe that comprises a PNA sequence that is complementary
to at least a portion of a target DNA sequence; b. attaching said PNA
probe to an electrode, said probe forming a self-assembled monolayer on
the electrode; c. hybridization of PNA probe with a sample dsDNA by
exposing the electrode with attached PNA probe to the dsDNA sample that
is in double-stranded form; d. exposing to an electroactive label the
hybrid of sample dsDNA with PNA probe attached onto the electrode; e.
subjecting the hybrid of PNA-dsDNA sample on the electrode interacted
with the electroactive label to an electrochemical method of measuring
the electroactive label signal indicative of the hybridization between
said PNA probe and sample dsDNA; f. comparing the electrochemical signal
of the hybrid of PNA-ds DNA sample to the electrochemical signal of said
PNA probe alone; and g. comparing the electrochemical signal of the
hybrid of PNA-ds DNA sample to the electrochemical signal of said PNA
probe and non-complementary dsDNA hybrid.

2. The method according to claim 1, wherein the PNA probe consists
essentially of the PNA sequence and a functional group, wherein said
functional group is linked to the PNA probe and also chemically binds to
the electrode.

3. The method according to claim 2, wherein the functional group that
chemically binds to the electrode is a thiol group.

4. The method according to claim 2, wherein the PNA probe forms a
self-assembled monolayer on the surface of a gold electrode, and wherein
said PNA probe attaches to the electrode by means of the functional
group.

5. The method according to claim 4, wherein the functional group is a
thiol group.

6. The method according to claim 1, wherein one strand of the dsDNA is
entirely complementary to the PNA probe, and wherein said complementary
sequence causes the formation of PNA-dsDNA hybrid.

7. The method according to claim 1, wherein the PNA-dsDNA hybrid
formation is monitored electrochemically following interaction of the
hybrid with an electrochemically active label, and wherein a measurement
of the electrochemical signal of the electrochemically active label that
is significantly different from the measurement of the electrochemical
signal of the PNA probe alone is indicative of hybrid formation.

8. The method according to claim 7, wherein the electrochemically active
label is Methylene blue.

9. The method according to claim 1, wherein sample dsDNA is discriminable
as being complementary or non-complementary to the probe PNA by comparing
the electrochemical signal of a mixture of the sample dsDNA and PNA probe
to the electrochemical signal of the probe PNA alone, wherein a
measurement of the electrochemical signal of said mixture that is
significantly different from the measurement of the electrochemical
signal of the PNA probe alone is indicative of the presence of
complementary dsDNA, and wherein a measurement of the electrochemical
signal of said mixture that is comparatively near to that of the probe
alone and significantly different from that of the PNA and complimentary
dsDNA hybride is indicative of the presence of non-complementary dsDNA.

10. A PNA-based method for the detection and discrimination of SNPs
directly on ds-oligonucleotides and PCR products, said method comprising
a. creating a PNA probe that comprises a PNA sequence that is
complementary to at least a portion of a target DNA sequence; b.
attaching said PNA probe to an electrode, said probe forming a
self-assembled monolayer on the electrode; c. hybridization of PNA probe
with a sample dsDNA by exposing the electrode with attached PNA probe to
the dsDNA sample which is in double-stranded form; d. exposing to an
electroactive label the hybrid of sample dsDNA with PNA probe attached
onto the electrode; e. subjecting the hybrid of PNA-dsDNA sample on the
electrode interacted with the electroactive label to an electrochemical
method of measuring the electroactive label signal indicative of the
hybridization between said PNA probe and sample dsDNA; f. comparing the
electrochemical signal of the hybrid of PNA-ds DNA sample to the
electrochemical signal of said PNA probe alone; and g. comparing the
electrochemical signal of the hybrid of PNA-ds DNA sample to the
electrochemical signal of said PNA probe and SNP (SBM) harboring dsDNA
hybrid.

11. The method according to claim 10, wherein the PNA probe is comprised
only of the PNA sequence and a functional group, wherein said functional
group is linked to the PNA probe and also chemically binds to the
electrode.

12. The method according to claim 11, wherein the functional group that
chemically binds to the electrode is a thiol group.

13. The method according to claim 10, wherein the PNA probe forms a
self-assembled monolayer on the surface of a gold electrode, and wherein
said PNA probe attaches to the electrode by means of the functional
group.

14. The method according to claim 10, wherein one strand of the dsDNA is
entirely complementary to the PNA probe, and wherein said complementary
sequence causes the formation of PNA-dsDNA hybrid molecule hybrid.

15. The method according to claim 10, wherein the PNA-dsDNA hybrid
formation is monitored electrochemically following interaction of the
hybrid with an electrochemically active label, and wherein a measurement
of the electrochemical signal of the electrochemically active label that
is significantly different from the measurement of the electrochemical
signal of the PNA probe alone is indicative of hybrid formation.

16. The method according to claim 15, wherein the electrochemically
active label is Methylene blue.

17. The method according to claim 10, wherein sample dsDNA is
discriminable as being complementary or SNP (SBM) harboring to the probe
PNA by comparing the electrochemical signal of a mixture of the sample
dsDNA and PNA probe to the electrochemical signal of the probe PNA alone,
and wherein a measurement of the electrochemical signal of said mixture
that is significantly different from the measurement of the
electrochemical signal of the PNA probe alone is indicative of the
presence of complementary dsDNA, and wherein a measurement of the
electrochemical signal of said mixture that is comparatively near to that
of the probe alone and significantly different from that of the PNA and
complementary dsDNA hybrid is indicative of the presence of SNP (SBM)
harboring dsDNA.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a method of detection and
discrimination of target DNA sequences and single nucleotide
polymorphisms (SNPs). More specifically, the present invention relates to
a method of detection and discrimination of target DNA sequences and SNPs
directly on double-stranded oligonucleotides or PCR products without
denaturing the target double-stranded oligonucleotides (ds
oligonucleotides) or PCR products, using PNA probes and based on
PNA-dsDNA hybrid formation.

DESCRIPTION OF RELATED ART

[0002] The detection of specific target DNA sequences is of increasing
importance in disease diagnostics, forensics, biotechnology, and many
other applications. DNA detection methods most commonly used include the
use of probe DNA, blot methods (such as the Southern blot), and
polymerase chain reaction (PCR).

[0003] Probe DNA can be used to detect a specific nucleic acid fragment.
In this method, a nucleic acid fragment is detected by a hybridization
reaction between the fragment and a probe DNA with a complementary
(according to the rules of Watson-Crick base pairing) sequence. A solid
carrier is commonly used on which to affix the probe DNA; this
carrier/probe DNA combination is known as a DNA chip. The carrier can be
made of a variety of substances, including glass, plastic, or an
electroconductive material (for example, see U.S. Pat. No. 6,713,255).
Basically, detection of DNA using a probe involves labeling a DNA
fragment with a detectable molecule (such as a fluorescent or
radioisotope label, or biotin), or the labeling of probe DNA with an
electrochemical label. Probe DNA that is complementary to the sequence of
the sample nucleic acid will bind to the sample, which is then detectable
using an applicable method (fluorometry, autoradiography, the
biotin-avidin method, voltammetry, etc.).

[0004] DNA blotting techniques involve the fractionalization of a sample
of DNA by gel electrophoresis. Once the DNA is on the gel, it is
denatured to create single-stranded DNA (ssDNA). The ssDNA is transferred
from the gel to a nitrocellulose membrane and fixed onto the membrane by
heating it in a vacuum, and is then incubated with a labeled ssDNA probe
that is complementary to a specific DNA sequence, and is thus capable of
hybridizing to any fragments in the sample having that sequence. Any
unbound probe is washed away, and bound probes are visualized using an
appropriate detection method (for example, see G. Karp, Cell and
Molecular Biology: Concepts and Experiments, 757, Fifth Ed., John Wiley &
Sons, Inc. (2008)).

[0005] PCR can also be used to detect target DNA. This method involves the
use of PCR oligonucleotide primers that are complementary to a target
nucleic acid sequence within a sample. These primers and other required
PCR ingredients are added to a mixture, and PCR is allowed to take place.
A PCR product will only be produced if the target DNA is present in the
sample (for example, see G. Karp, Cell and Molecular Biology: Concepts
and Experiments, 765, Fifth Ed., John Wiley & Sons, Inc. (2008)).

[0006] The detection of single nucleotide polymorphisms (SNPs) is also of
increasing scientific, medical, and pharmaceutical importance. SNPs are
discrepancies in the DNA sequence between two individuals of the same
species. SNPs are not only implicated in disease development, but also
may be part of the reason why individuals react differently to the same
pharmaceutical drugs (for example, see SNPs: Variations on a Theme,
http://www.ncbi.nih.gov/About/primer/snps.html). Hybridization methods
(such as dynamic allele-specific hybridization (DASH), use of molecular
beacons, and SNP microarrays) are commonly used to find these small
changes. Enzyme based methods (such as restriction fragment length
polymorphism (RFLP), use of Flap endonuclease ("invader assay"), use of
5'-nuclease ("Taqman assay"), and oligonucleotide ligase assay) are also
used (for example, see R. Rapley & S. Harbron, Molecular Analysis and
Genome Discovery, 42-59, John Wiley & Sons, Ltd. (2004)).

[0007] In addition to using DNA nucleic acid as a probe for the detection
of target DNA sequences and SNPs, peptide nucleic acid (PNA) is also
usable in certain applications. PNA is a synthetic analogue of DNA in
which the DNA-type phosphate sugar backbone is replaced by a peptide
backbone. Like biological nucleic acids, PNA can hybridize to DNA and RNA
with sequence specificity; however, because PNA is not a naturally
occurring compound, it is not susceptible to enzymatic degradation and
has a longer shelf life (for example, see U.S. Pat. No. 6,280,946).
Additionally, PNA's lack of a charge causes PNA/DNA to hybridize more
readily and with greater strength than DNA/DNA (for example, see E. Ortiz
et al., Cellular Probes, vol. 12, pp. 219-226 (1998)). This higher
affinity allows significantly shorter PNA oligomers to be used in
probe-based analyses as opposed to the 25- to 30-unit length typical for
an oligonucleotide probe required to obtain a stable hybrid (for example,
see U.S. Pat. No. 5,985,563).

[0008] PNAs have a higher thermal instability of mismatching bases than
DNA/DNA combinations because PNAs exhibit a greater specificity for their
complementary nucleic acids than do traditionally used nucleic acid
probes of corresponding sequence (for example, see U.S. Pat. No.
5,985,563).

[0009] PNA has been used in several nucleic acid detection techniques,
such as: (1) use in a PNA chip used for highly sensitive qualitative
analysis of a nucleic acid fragment complementary to the chip's PNA
fragment (U.S. Pat. No. 6,713,255); (2) PNA probes for the universal
detection of bacteria and/or eucarya (U.S. Pat. No. 6,280,946); (3) PNA
probes for the detection of ribosomal RNA (U.S. Pat. No. 5,985,563); and
(4) use in molecular beacons for rapid detection of PCR amplicons (E.
Ortiz et al. (1998)). PNA is also used, for example, in (5) enhancing
amplification in PCR (U.S. Pat. No. 5,656,461); and (6) inclusion in a
PNA-DNA-PNA chimeric macromolecule that has increased nuclease
resistance, increased binding affinity to a complementary nucleic acid
strand, and that activates RNase H enzyme (U.S. Pat. No. 5,700,922).

[0010] As one of ordinary skill in this art understands, however, is that
all of these DNA- and SNP-detection techniques and PNA applications have
in common the requirement that, at some point in the process, target
double-stranded DNA (dsDNA) be denatured into ssDNA. In fact, nucleic
acid hybridization is based on the phenomenon that complementary strands
of nucleic acids from different sources can anneal to each other and
create a hybrid double-stranded molecule. Nucleic acid hybridization is
the driving force behind the use of DNA probes, blotting techniques, and
PCR.

[0011] The prior art, although teaching the highly stable and specific
binding of PNA to DNA, also requires the denaturation of dsDNA into
ssDNA. In other words, PNA binds to ssDNA to create a double-stranded
detectable hybrid. Denaturation of DNA represents a time-consuming step
in nucleic acid detection, and can also provide the opportunity for
machine or human error in a procedure.

SUMMARY OF THE INVENTION

[0012] It is thus the purpose of the present invention to provide a more
streamlined, simplified method for the detection of target DNA sequences
and SNPs that may greatly reduce the assay time and the number of
required procedures in the detection protocol.

[0013] The present invention achieves these goals by providing a method
for the detection and discrimination of target DNA sequences and SNPs
directly on a double-stranded oligonucleotide (ds-oligonucleotide) chain
or PCR product through the use of PNA-dsDNA hybrid formation.

[0014] Generally, this invention relates to a method of using the
PNA-dsDNA hybrid to find a target DNA sequence within a sample of DNA.
The invention also relates to a method of using the PNA-dsDNA hybrid to
discriminate between DNA sequences within a sample that are complementary
to the PNA probe, and sequences that are either non-complementary or
contain SNPs.

[0015] In one embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of target DNA sequences directly on ds-oligonucleotides.
This method uses a PNA probe that is complementary to a target DNA
sequence and thus forms a PNA-dsDNA hybrid with the ds-oligonucleotide.

[0016] In another embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of single nucleotide polymorphisms (SNPs) within DNA
sequences directly on ds-oligonucleotides. This method uses a PNA probe
that is complementary to a target DNA sequence and thus forms a PNA-dsDNA
hybrid with the ds-oligonucleotide.

[0017] In another embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of target DNA sequences directly on double-stranded PCR
products. This method uses a PNA probe that is complementary to a target
DNA sequence and thus forms a PNA-dsDNA hybrid with the double-stranded
PCR product.

[0018] In another embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of single nucleotide polymorphisms (SNPs) within DNA
sequences directly on double-stranded PCR products. This method uses a
PNA probe that is complementary to a target DNA sequence and thus forms a
PNA-dsDNA hybrid with the double-stranded PCR product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention is illustrated by way of example and not by
limitation in the accompanying drawings, in which:

[0020] FIG. 1

shows the differential pulse voltammograms for the reduction signal of MB
following self-assembly of 100 .mu.M of the PNA probe on the gold
electrode (AuE) for different time durations including: 30 minutes (curve
a), 1 hour (curve b), 7 hours (curve c), overnight (curve d), and 3 days
(curve e);

[0021] FIG. 2

shows the differential pulse voltammograms for the reduction signal of MB
following self-assembly of the 13-mer PNA probe on the AuE (curve a),
after the hybridization with the complementary UGT ds-oligonucleotide
(curve b), after the interaction with the non-complementary IL-2
ds-oligonucleotide (curve c), and after the interaction with the UGT.SBM
ds-oligonucleotide containing one base mismatch (curio d);

[0022] FIG. 3

shows the results of agarose gel electrophoresis of PCR products compared
to 100 bp (A) and 1 kb (B and C) DNA ladders. PCR amplicons are of the
following: complementary UGT1A9 with a length of 166 bp (A1), the
non-complementary internal transcribed sequence (ITS) with a length of
773 bp (B1), IL-10 (C1) and IL-4 (C2) promoter regions with lengths of
1058 and 1047, respectively, and the mutant UGT1A9.SBM containing one
base mismatch with a length of 166 bp (A2); and

[0023] FIG. 4

shows differential pulse voltammograms for the reduction signal of MB
following self-assembly of the 13-mer PNA probe on the AuE (curve a),
after hybridization with the complementary UGT1A9 PCR sample (curve b),
after interaction with the non-complementary IL-4, ITS, and IL-10 PCR
products (curves c through e, respectively), and after interaction with
the mutant UGT1A9.SBM PCR product (curve f). All PCR products were used
without denaturation of the dsDNA into ssDNAs.

DETAILED DESCRIPTION THE INVENTION

Definitions

[0024] "PNA" or "peptide nucleic acid" is used herein to mean any
oligomer, linked polymer or chimeric oligomer, comprising two or more PNA
subunits (residues), with a peptide backbone comprised of repeating
N-(2-aminoethyl)-glycine units linked by peptide bonds. This definition
also includes any of the compounds referred to or claimed as peptide
nucleic acids in U.S. Pat. Nos. 5,539,082; 5,527,675; 5,623,049;
5,714,331; 5,736,336; 5,773,571; or 5,786,461 (all of which are herein
incorporated by reference). The term "peptide nucleic acid" or "PNA"
shall also apply to polymers comprising two or more subunits of those
nucleic acid mimics described in the following publications: Diderichsen
et al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem.
Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:
687-690 (1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995);
Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Lowe et
al., J. Chem. Soc. Perkin Trans. 1: 539-546 (1997); Lowe et al., J. Chem.
Soc. Perkin Trans. 1: 555-560 (1997); Petersen et al., Bioorg. Med. Chem.
Lett. 6: 793-796 (1996); Deiderichsen, U., Bioorganic & Med. Chem. Lett.
8: 165-168 (1998); Cantin et al., Tett. Lett. 38: 4211-4214 (1997);
Ciapetti et al., Tetrahedron 53: 1167-1176 (1997); Lagriffoule et al.,
Chem. Eur. J. 3: 912-919 (1997); and WIPO patent application WO96/04000
(Shah et al., "Peptide-based nucleic acid mimics (PENAMs)"). "PNA probe"
is used herein to mean a PNA sequence that is complementary to at least a
portion of a target DNA sequence. The PNA probe may also include a
thiolated head group and a linker segment, but does not include any tag,
label, or marker used for detection of the probe using radioactive
detection, fluorescence, or any other detection methods. "Target DNA" or
"target sequence" is used herein to mean the nucleic acid sequence of
sample DNA to be detected in an assay and to which at least a portion of
the probing PNA sequence is designed to hybridize. "PNA-dsDNA" or
"PVA-dsDNA hybrid" is used herein to mean the pairing of PNA and
double-stranded DNA, which results in a multiple-stranded hybrid.
"Double-stranded oligonucleotide" or "ds-oligonucleotide" is used herein
to mean a short synthetic polymer comprised of two complementary strands
of at least two nucleotides. "Self-assembled monolayer" or "SAM" is used
herein to mean a self-organized layer of amphiphilic molecules, such
molecules comprising a head group with a particular affinity for the
surface of a specific substrate, and a tail containing any of a variety
of functional groups. "Single base mutation" or "SBM" is used herein to
mean a DNA sequence variation in which a single base pair is substituted
(changed), added (inserted), or deleted (removed), and which is either
naturally occurring or artificially induced. "Single nucleotide
polymorphism" or "SNP" is used herein to mean a discrepancy in the DNA
sequence in at least one base pair between two individuals of the same
species. The terms of SNP and SBM have the same meaning in this art.

DESCRIPTION OF THE INVENTION

[0025] The present invention allows for the detection and selective
discrimination of target DNA sequences directly on ds-oligonucleotides
and double-stranded PCR products. Furthermore, the detection of DNA
single base mutations on ds-oligonucleotides and double-stranded PCR
products is also taught. Detection procedure is carried out without the
need for denaturation of a dsDNA sample into ssDNA. This strategy is a
label- and enzyme-free method that relies on direct interaction of a PNA
probe with the target dsDNA. Accordingly, it is a simplified and rapid
DNA and mutation detection protocol. The method could be considered as a
fundamental platform for the development of further DNA detection
techniques based on PNA-dsDNA hybridization.

[0026] It has been discovered that PNA can form not only a PNA-ssDNA
duplex, as seen in traditional hybridization methods, but can also form
other structures including a PNA-dsDNA hybrid for example, a PNA-dsDNA
triplex hybrid) when pyrimidine or purine bases of the PNA fit into the
major groove of the DNA double helix. Generally, two homopyrimidine PNA
molecules displace the duplex DNA pyrimidine strand and form a hybrid
with the purine DNA strand (such structure commonly called a "P-loop")
(for example, see M. D. Frank-Kamenetskii et al., Annu. Rev. Biochem.,
vol. 64, pp. 64-95 (1995)). It is no longer required that the
ds-oligonucleotide and PCR product be denatured into a ssDNA in order to
detect a target DNA sequence and this hybrid is used as a method to
detect target DNA sequences directly on the ds-oligonucleotides and
double-stranded PCR products without denaturing the target dsDNA into
ssDNA.

[0027] Additionally, sequence mutations may also be detected without
denaturing the target DNA. This is based on the observation that,
although PNA has a high binding affinity for complementary DNA, it binds
less tenuously to a mismatched DNA sequence than two non-complementary
DNA sequences would bind to each other. Therefore, a low occurrence of
binding between a PNA probe and a target DNA sequence indicates that a
SNP exists in the sample DNA sequence, as opposed to the high occurrence
of binding that is expected between a PNA probe and complementary DNA
sequence. Furthermore, because of the ability to use a short fragment of
PNA as an effective probe, mutations can be isolated more specifically in
a given DNA sequence.

[0028] The PNA probe method of this invention is particularly useful for
the detection and discrimination of target DNA sequences and SNPs
directly on ds-oligonucleotides and PCR products from clinical specimens.
There is an increased recognition of the importance of SNPs in disease
development, and the method disclosed herein provides an effective way of
detecting these mutations.

[0029] This invention broadly comprises a PNA-based electroactive
label-mediated electrochemical method for the detection and
discrimination of a target DNA sequence and SNP directly on dsDNA using a
PNA probe, using the PNA-dsDNA hybrid formation that occurs between the
dsDNA and PNA probe.

[0030] More specifically, the above-described method comprises the use of
a PNA probe that has not been coupled with any marker or label, and that
contains only a functional group (here, a thiol group provided by
cysteine) that is connected to the probe with a linker segment (here,
8-amino-3,6-dioxa-octanoic acid).

[0031] The above-described method also comprises the use of a gold
electrode (AuE), onto which the thiolated PNA probe attaches (via the
thiol group), resulting in the formation of a self-assembled monolayer
(SAM).

[0032] The AuE/SAM is then exposed to a sample of DNA. If the sample
contains the target DNA sequence, results in the formation of a stable
PNA-dsDNA hybrid resulting from the interaction of the PNA probe with the
dsDNA, one strand of which is completely complementary to the probe.

[0033] Monitoring of the hybrid formation can be achieved using an
electrochemical approach. In a preferred embodiment, the formation of the
PNA-dsDNA hybrid is monitored electrochemically following interaction of
the hybrid with an electroactive label such as Methylene blue (MB).
Neither the PNA nor target DNA sequences are tagged, labeled, or marked
with any tag, label, enzyme or marker prior to this step; it is only
after mixing the PNA probe and dsDNA sample that MB is added. The
preferred electrochemical method for monitoring hybrid formation is
differential pulse voltammetry (DPV).

PREFERRED EMBODIMENTS

[0034] In one embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of target DNA sequences directly on ds-oligonucleotides.
This method uses a PNA probe that is complementary to a target DNA
sequence and thus forms a PNA-dsDNA hybrid with the ds-oligonucleotide.

[0035] In another embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of single nucleotide polymorphisms (SNPs) within DNA
sequences directly on ds-oligonucleotides. This method uses a PNA probe
that is complementary to a target DNA sequence and thus forms a PNA-dsDNA
hybrid with the ds-oligonucleotide.

[0036] In another embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of target DNA sequences directly on double-stranded PCR
products. This method uses a PNA probe that is complementary to a target
DNA sequence and thus forms a PNA-dsDNA hybrid with the double-stranded
PCR product.

[0037] In another embodiment, this invention relates to a PNA-based
electroactive-label-mediated electrochemical method for the detection and
discrimination of single nucleotide polymorphisms (SNPs) within DNA
sequences directly on double-stranded PCR products. This method uses a
PNA probe that is complementary to a target DNA sequence and thus forms a
PNA-dsDNA hybrid with the double-stranded PCR product.

[0038] All electrochemical experiments were performed in a 5 ml volume
cell, connected to an AUTOLAB PGSTAT 30 electrochemical analysis system
and GPES 4.9 software package (Eco Chemie., the Netherlands). This
three-electrode system was composed of a gold disk electrode (having a
diameter of 3 mm, Bioanalytical Systems (BAS), West Lafayette, Ind.), a
saturated calomel electrode (SCE) as the reference electrode, and a
platinum wire as the auxiliary electrode. Supporting electrolyte was
deoxygenated by purging with nitrogen gas for 10 minutes prior to
measurements, and the cell was blanketed with nitrogen for the duration
of the experiments. All of the electrochemical experiments were carried
out at laboratory ambient temperature.

Preparation of the Working Electrode

[0039] A gold disk electrode (having a diameter of 3 mm, Bioanalytical
Systems (BAS), West Lafayette, Ind.) was used for all the experiments.
The electrode was polished with wet alumina slurry (Alpha and Gamma
Micropolish II deagglomerated alumina, Buehler, Lake Bluff, Ill.) on a
felt pad for at least 10 minutes and rinsed repeatedly with water. The
polished electrode was then dipped in a solution of 1:1 EtOH--H.sub.2O
containing 0.5 M NaBH.sub.4 for about 20 minutes. The electrode was then
treated in 0.05 M H.sub.2SO.sub.4 with a cycling of the potential between
-0.3 and 1.5 V with a scan rate of 0.1 Vs-1 until the cyclic voltammogram
did not change (indicating that the electrode surface was clean).
Finally, the electrode was rinsed with water before probe self-assembly.

[0040] PNA self-assembly onto the surface was performed as follows: The
electrode was placed upside down in a disposable glass tube. An aliquot
of 5 .mu.L of the 50 .mu.M PNA probe was deposited onto the gold
electrode. The tube was sealed to prevent water evaporation and left for
self-assembly at room temperature overnight. The electrode was then
rinsed with water and dipped in an aqueous 1 mM 6-mercapto-1-hexanol
(MCH) solution at room temperature for 30 minutes. MCH deposition is
important both to competitively remove non-covalently adsorbed PNA
molecules and to fill "pinholes" within the PNA monolayer. The electrode
was washed repeatedly with water before every use.

Evaluation of PNA Self-Assembled Monolayer Formation

[0041] The extent of probe self-assembly was monitored by the probe
influence on the electrochemical signal of
Fe(CN).sub.6.sup.3-/Fe(CN).sub.6.sup.4- system on the pretreated AuE
based on the decline in the height of the
Fe(CN).sub.6.sup.3-/Fe(CN).sub.6.sup.4- redox peaks. For this purpose,
the electrode was scanned between -0.5 and 0.6 V in 10 mM
K.sub.3Fe(CN).sub.6+10 mM K.sub.4Fe(CN).sub.6 solution.

PNA-dsDNA Hybridization

[0042] Hybridization between the probe and PCR products was carried out
following gel extraction of the desired DNA bands from agarose gel using
QIAquick Purification Kit (QIAGEN, Germany). The extracted DNA fragments
were used for the hybridization without denaturation (i.e. as a
double-stranded structure).

[0043] Hybridization between the PNA probe and ds-oligonucleotides or
double-stranded PCR products was performed using the direct dropletting
method, the same protocol used for the probe self-assembly on the
electrode. A 5 .mu.L droplet of ds-DNA sample was deposited onto the gold
electrode. The tube was sealed to prevent water evaporation and kept at
room temperature during overnight hybridization, resulting in the
formation of the PNA-dsDNA hybrid. The PNA-dsDNA/AuE was rinsed with
water.

[0044] The target DNA molecules were all used in their stock concentration
(100 .mu.M) as a 5 .mu.L drop.

Label Binding to the Hybrid

[0045] Methylene blue was accumulated on the PNA self-assembled AuE by
immersing the modified electrode into the 20 mM Tris-HCl buffer (pH 7.0)
containing 20 .mu.M MB and 20 mM NaCl for 5 minutes with 200 rpm stirring
without applying any potential to this electrode. Once MB was
accumulated, the electrode was rinsed with 20 mM Tris-HCl buffer (pH 7.0)
for 20 seconds. The same protocol was applied for the accumulation of MB
on the bare electrode and probe-modified electrodes following the
hybridization with dsDNA molecules.

Voltammetric Measurements

[0046] The reduction signal of the accumulated MB was measured by using
differential pulse voltammetry (DPV) in 20 mM Tris-HCl buffer (pH 7.0)
containing 20 mM NaCl and scanning the electrode potential between 0.20
and -0.60 V at a pulse amplitude of 25 mV. The raw data were treated
using the Savitzky and Golay filter (level 2) of the GPES software,
followed by the GPES software moving average baseline correction using a
"peak width" of 0.01.

Preliminary Investigation

[0047] Once the SAM formation of the PNA probe on the AuE was confirmed
using the Fe(CN).sub.6.sup.3-/Fe(CN).sub.6.sup.4- redox system, SAM
formation and time and probe concentration variables, which affect the
performance of the sensor, were studied and special emphasis was given to
the optimization of these experimental variables.

[0048] In order to obtain the optimum self-assembly time, self-assembly of
the PNA on the AuE was conducted for different time durations. FIG. 1
shows the differential pulse voltammograms for the reduction signal of MB
following self-assembly of 100 .mu.M of the PNA probe on the AuE for
different times including: 30 minutes (curve a), 1 hour (curve b), 7
hours (curve c), overnight (curve d), and 3 days (curve e). As seen in
FIG. 1, the MB reduction signal elevated as the self-assembly time
increased to about overnight and maintained constant for 3 days.
Therefore, overnight incubation was suggested as optimum time for
self-assembly of PNA on the AuE.

[0049] The effect of PNA concentration on the self-assembly of the PNA on
the electrode was studied using DPV measurements of MB signals following
self-assembly of the PNA on the AuE using PNA solutions of 10 .mu.M, 25
.mu.M, 50 .mu.M, and 100 .mu.M. The results obtained from the
Voltammetric measurements revealed that the MB reduction signal elevated
as the PNA concentration increased (104.8.+-.16.7 nA, 181.5.+-.9.9 nA,
258.2.+-.14.9 nA, and 359.1.+-.22.4 nA for 10, 25, 50, and 100 .mu.M PNA
concentration, respectively). Therefore, 50 .mu.M was determined to be a
proper and moderate PNA concentration for self-assembly of the probe on
the AuE.

[0050] The following examples demonstrate in greater detail the procedures
that can be used in the present invention. The DNA and PNA sequences used
herein are, however, only suggestive, and are not intended to limit the
scope of the invention in any way. One skilled in the art understands how
to substitute and/or adapt the PNA sequence to investigate any desired
target DNA sequence or SNP.

[0052] A 13-mer PNA oligonucleotide (P275) corresponding to the sense
strand of human uridin diphosphate glucuronosyltransferase 1A9 (UGT1A9)
gene promoter region was used as the probe. P275 had a sequence of
Cys-OO-AGT TTA GTA GCA G, where "cys" was cysteine amino acid and "O" was
an 8-amino-3,6-dioxa-octanoic acid linker. The cysteine at the N-terminal
of the probe provided a thiol group that allowed covalent binding of the
probe to the electrode surface, and the "O" linker separated the
hybridization segment from the thiolated terminal. P275 covered 13
nucleotides of the promoter region of the UGT1A9 gene, from nucleotides
-280 to -267 from the start codon including (A/T) SNP at position -275.

[0053] Two DNA oligonucleotides were used for preparation of the
complementary

[0054] UGT ds-oligonucleotide: DC275 (5'-CTG CTA CTA AAC T-3'), which was
complementary to the probe; and D275 (5'-AGT TTA GTA GCA G-3'), which was
identical to the probe.

[0056] The following DNA oligonucleotides were used for preparation of the
UGT.SBM ds-oligonucleotide containing a single base mismatch with the PNA
probe: DMM275 (5' ACT TTA GAA GCA G-3'), which was identical to the probe
with a single base substitution (T/A) at nucleotide 8; and DCMM275
(5'-CTG OTT CTA AAC T-3'), which was complementary to the probe with a
single nucleotide mismatch (A/T) at the corresponding position;
nucleotide 6 in this oligonucleotide.

[0058] Other chemicals were of analytical reagent grade. Distilled,
deionized, and sterilized water was used in all solution preparation. All
DNA solutions were kept frozen at -20.degree. C. and all the experiments
were performed at room temperature in an electrochemical cell.

Preparation of Double-Stranded Oligonucleotides

[0059] To prepare ds-oligonucleotides, equal concentrations (50 .mu.M) of
pair oligonucleotides were effectively mixed in a microtube. The mixed
solution was then heated at 95.degree. C. in a water bath for 10 minutes,
and was then allowed to cool down gradually to room temperature.

(1) Human Genomic DNA Extraction

[0060] Human genomic DNA was extracted from a peripheral blood sample
using a high-yield DNA purification kit (DNP.TM. Kit, CinnaGen, Inc.).
Two hundred (200) .mu.L of the sample blood was centrifuged at 3000 rpm
for 10 minutes, from which the serum was then separated. The precipitated
cells were suspended in lysis solution and lysed by incubation at
37.degree. C. for 20 minutes. Precipitation solution was then added and
the tube was placed at 20.degree. C. for another 20 minutes. The solution
was centrifuged at 14000 rpm for 20 minutes at 4.degree. C., and then the
supernatant was poured off. The DNA pellet was washed with wash buffer
and dried. Extracted DNA was finally dissolved in solvent buffer and kept
frozen at -20.degree. C.

(2) Plant Genomic DNA Extraction

[0061] Genomic DNA extraction from Thymus pubescens was carried out
according to the CTAB protocol described in J. J. Doyle et al.,
"Isolation of Plant DNA from Fresh Tissue," Focus vol. 12, pp. 13-15
(1990). Accordingly, leaves were immersed in liquid nitrogen and then
ground into fine powder using a mortal and pestle. The powdered leaves
were lysed by addition of a lysis solution (Tris-HCl 10 mM (pH 8.0), EDTA
1 mM, 1.4 M NaCl, 2% CTAB and 1% PVP) and incubation at 65.degree. C. for
20 minutes. Genomic DNA extraction was carried out with equal volume of
chloroform-isoamylalcohol (24:1) and precipitated from the aqueous phase
with 0.6 volume (v/v) isopropanol. The extracted DNA was further purified
using ethanol 70%.

[0063] In order to amplify the mutant form of the UGT1A9 (UGT1A9.SBM) DNA
fragment, containing a single base mutation, UGTF and UGTRa (5'-TGC AAT
GTT AAG TTT AGA AGC AG-3') oligonucleotides were used as forward and
reverse primers, respectively. It is noted that UGTRt and UGTRa have the
same sequence with a single nucleotide substitution (T/A) at position 18.

[0064] PCR reactions contained about 100 ng template DNA, 0.1 .mu.M of
each primer, 1 mM of deoxynucleoside triphosphates (dNTPs), 1.5 mM
MgCl.sub.2, and 1 .mu.l Taq DNA polymerase in a total volume of 50 .mu.L.
The amplification program consisted of the following steps:

[0071] PCR amplification of mutant UGT1A9 (UGT1A9.SBM) was carried out
with the same program except that the annealing temperature was reduced
to 54.degree. C.

[0072] The PCR products were separated on agarose gel and DNA bands were
extracted from the gel using QIAquick PCR Purification Kit (QIAGEN,
Germany). The pair primers UGTF/UGTRa and UGTF/UGTRt cover the UGT1A9
promoter region from nucleotide -426 to -758 from the start codon.

[0090] PCR amplification of the IL-10 promoter region was performed in the
same manner as the IL-4 promoter region amplification.

Example 2

Evaluations of ds-Oligonucleotide Using PNA Probe

Preparations

[0091] This example builds from the preparations described in Example 1.

[0092] The 13-mer PNA oligomer was self-assembled on the AuE as the probe
and was hybridized with the complementary UGT ds-oligonucleotide. FIG. 2
shows the differential pulse voltammograms for the reduction signal of MB
following self-assembly of the probe on the AuE (curve a) and after the
hybridization of the probe with the complementary dsDNA (curve b). The
self-assembled probe displayed a current of 258.2.+-.14.9 nA following
interaction with MB. However, a significant increase in the MB signal was
observed following the hybridization of the PNA probe with the
complementary ds-oligonucleotide (from 258.2.+-.14.9 nA to 671.3.+-.36.5
nA). The observed rise in MB signal is attributable to the intercalation
of MB within the PNA-dsDNA structure as an intercalator. Therefore, the
elevation in the MB signal is attributed to the hybridization of the PNA
probe with the complementary ds-oligonucleotide on the electrode surface
and consequently may be used for detection of a target DNA sequence.

Selectivity Study for ds-Oligonucleotide Detection

[0093] The hybridization of the PNA probe with the non-complementary dsDNA
was carried out to assess selectivity of the proposed approach. For this
purpose, IL-2 ds-oligonucleotide was prepared as a non-complementary
ds-oligonucleotide and used for the selectivity study. Curve c in FIG. 2
shows the DPV for the reduction signal of MB after interaction of the PNA
modified electrode with the non-complementary ds-oligonucleotide. The
interaction between the non-complementary ds-oligonucleotide and the
probe-modified electrode did not lead to a significant increase in the MB
signal (279.1.+-.20.6 nA), and the signal was nearly equal to that of the
probe. This result indicates that only the complementary
ds-oligonucleotide could form an entirely matched hybridized structure
with the probe that results in the significant increase in the
accumulation of MB.

[0094] A series of three repetitive measurements led to reproducible
results and the relative standard deviation was 7.38% for the
non-complementary sequence detection on ds-oligonucleotide. These results
clearly demonstrate the feasibility of detection and discrimination of a
target DNA sequence on a ds-oligonucleotide.

Single Base Mismatch Detection on ds-Oligonucleotide

[0095] The effective discrimination against a single base mismatch (SBM)
directly on ds-oligonucleotide was investigated using UGT.SBM
ds-oligonucleotide containing a single base mismatch (mutation). Curve d
in FIG. 2 shows the DPV for the reduction signal of MB after interaction
of UGT.SNP ds-oligonucleotide with the PNA-modified electrode. As shown,
in the presence of UGT.SBM ds-oligonucleotide, the MB signal was 320
8.+-.27.5 nA. This current is significantly lower than the MB signal of
the complementary ds-oligonucleotide due to the lack of entire
hybridization between the probe and the single-base mismatched
ds-oligonucleotide. The relative standard deviation was 8.57% for the
detection of single base mismatch on the ds-oligonucleotide.

[0097] PCR amplification of the complementary UGT1A9 DNA fragment was
conducted and followed by agarose gel electrophoresis. Electrophoresis of
the complementary UGT1A9 PCR sample showed a DNA band with a length of
166 bp, indicating the presence of the complementary UGT1A9 DNA amplicon
in the PCR sample (FIG. 3A).

[0098] FIG. 4 shows typical ADP voltammograms for the PNA probe-modified
AuE before (curve a) and after (curve b) the hybridization with the
complementary PCR sample. As illustrated, following the hybridization of
the complementary PCR sample with the probe, the MB signal increased
remarkably from 258.2.+-.14.9 nA to 988.4.+-.73.5 nA. The observed raise
in the MB signal is referred to the formation of the PNA-dsDNA structure
and consequently is used for the detection of a target dsDNA sequence on
PCR product.

Selectivity Study for Double-Stranded PCR Product

[0099] Hybridization experiments with the non-complementary PCR products
were carried out to assess whether the suggested DNA detection strategy
responds selectively to the target DNA sequence. For this purpose, three
different non-complementary PCR products, including human interleukin-4
and interleukin-10 promoter regions and Thymus pubescens internal
transcribed spacer (ITS) region, were used. The DNA fragments were
amplified using PCR technique and the presence of DNA bands with lengths
657, 1047, and 1138 bps, respectively, in agarose gel electophoresis it
the to amplification of the non-complementary PCR samples indicated
(IL-4, IL-10, and ITS spacer region).

[0100] Curves c through e in FIG. 4 display the differential pulse
voltammograms of the MB reduction signal after interaction of the
probe-modified electrode with the non-complementary PCR products. Upon
the hybridization of ITS dsDNA, a subsequent decrease was observed in the
MB current compared to that of the complementary UGT1A9 double-stranded
PCR product from 988.4.+-.73.5 nA to 308.5.+-.27.6 nA. Similar results
were obtained for the non-complementary IL-4 and IL-10 samples whose MB
signals were 283.2.+-.25.7 and 314.6.+-.43.1 nA, respectively.

Detection of Single Base Mutation on Double-Stranded PCR Product

[0101] The 166-bp UGT1A9.SBM DNA fragment containing a single base
mutation was amplified using PCR (FIG. 3A) and hybridized with the PNA
probe following gel extraction. The interaction between UGT1A9.SBM PCR
product and the probe led to an MB signal current of 371.2.+-.30.8 nA
(FIG. 4, curve f), representing a significant difference between this
current and that of the complementary UGT1A9 (988.4.+-.73.5 nA). This
observation clearly confirmed the ability of the developed strategy to
detect and discriminate the DNA mutation directly on double-stranded PCR
product.

Because the above-mentioned techniques show that the interaction of a PNA
probe with a double-stranded DNA (ds-oligonucleotide or PCR product)
sample can be useful for detecting a target sequence or SNP without
requiring dsDNA denaturation into ssDNA, we claim the following: